Myosin X is an unconventional myosin with a conserved motor domain followed by three IQ motifs that are potential light chain binding domains. There is a short segment predicted to form a coiled-coil that probably allows for dimerization to produce a two-headed molecule. Little is known about the function of myosin X in cells and it appears to be expressed at low abundance. In order to characterize the in vitro enzymatic properties of this myosin, we have engineered fragments corresponding to an HMM and S1 of bovine myosin X for expression in Sf9 cells. Initially, we coexpressed these fragments with calmodulin. Both constructs yielded soluble myosin containing bound calmodulin that was purified by FLAG-affinity chromatography. The myosin X-HMM-like fragment binds actin in an ATP-dependent manner and has an actin-activated MgATPase with a Vmax of 10 per sec and a Km of 5 micromolar at 37C. The MgATPase is relatively ionic strength insensitive compared to conventional myosin IIs. The myosin X-HMM translocates actin filaments in a rate of 0.18+0.05 microns/s in the in vitro motility assay. Motility requires moderate densities of myosin bound to the surface and is enhanced by the presence of methylcellulose, a viscosity inducing reagent. Thus, myosin X does not appear to be a candidate for a processive vesicle transporter. The S1 fragment was also enzymatically active in solution, but did not support in vitro motility. It will be used to examine the transient kinetics. Recently, we have also co-expressed the myosin X heavy chain fragments with a calmodulin-like protein (termed CLIP). CLIP binds to the heavy chain and supports activity. The affinity for CLIP appears to be similar to that of calmodulin. We have attempted to immunoprecipitate myosin X from cells in order to identify the endogenous light chains, but have not been successful. Finally, we have expressed full length myosin X in baculovirus, but find that there is proteolysis occurring within the cells which probably results in a considerable amount of single-headed myosin being produced.